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1.  Single cell antimicrobial susceptibility testing by confined microchannels and electrokinetic loading 
Analytical chemistry  2013;85(8):3971-3976.
Multidrug-resistant pathogens are an emerging global health problem. In addition to the need of developing new antibiotics in the pipeline, the ability to rapidly determine the antibiotic resistance profiles of bacteria represents one of the most crucial steps toward the management of infectious diseases and the prevention of multidrug-resistant pathogens. Here, we report a single cell antimicrobial susceptibility testing (AST) approach for rapid determination of the antibiotic resistance of bacterial pathogens. By confining individual bacteria in gas permeable microchannels with dimensions comparable to a single bacterium, the antibiotic resistance of the bacteria can be monitored in real-time at the single cell level. To facilitate the dynamic loading of the bacteria into the confined microchannels for observation, AC electrokinetics is demonstrated for capturing bacteria to defined locations in high-conductivity AST buffer. The electrokinetic technique achieves a loading efficiency of about 75% with a negligible effect on the bacterial growth rate. To optimize the protocol for single cell AST, the bacterial growth rate of individual bacteria under different antibiotic conditions has been determined systematically. The applicability of single cell AST is demonstrated by the rapid determination of the antimicrobial resistant profiles of uropathogenic clinical isolates in Mueller-Hinton media and in urine. The antibiotic resistance profiles of bacteria can be determined in less than one hour compared to days in standard culture-based AST techniques.
doi:10.1021/ac4004248
PMCID: PMC4102406  PMID: 23445209
2.  Plasma Lithography Surface Patterning for Creation of Cell Networks 
Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.
doi:10.3791/3115
PMCID: PMC3197071  PMID: 21694697
3.  Electrokinetic Stringency Control in Self-Assembled Monolayer-based Biosensors for Multiplex Urinary Tract Infection Diagnosis 
Nanomedicine : nanotechnology, biology, and medicine  2013;10(1):10.1016/j.nano.2013.07.006.
Rapid detection of bacterial pathogens is critical toward judicious management of infectious diseases. Herein, we demonstrate an in situ electrokinetic stringency control approach for a self-assembled monolayer-based electrochemical biosensor toward urinary tract infection diagnosis. The in situ electrokinetic stringency control technique generates Joule heating induced temperature rise and electrothermal fluid motion directly on the sensor to improve its performance for detecting bacterial 16S rRNA, a phylogenetic biomarker. The dependence of the hybridization efficiency reveals that in situ electrokinetic stringency control is capable of discriminating single-base mismatches. With electrokinetic stringency control, the background noise due to the matrix effects of clinical urine samples can be reduced by 60%. The applicability of the system is demonstrated by multiplex detection of three uropathogenic clinical isolates with similar 16S rRNA sequences. The results demonstrate that electrokinetic stringency control can significantly improve the signal-to-noise ratio of the biosensor for multiplex urinary tract infection diagnosis.
doi:10.1016/j.nano.2013.07.006
PMCID: PMC3858494  PMID: 23891989
Urinary tract infection; Stringency control; Multiplex detection; Matrix effects; Self-assembled monolayers
4.  Probing Mechanoregulation of Neuronal Differentiation by Plasma Lithography Patterned Elastomeric Substrates 
Scientific Reports  2014;4:6965.
Cells sense and interpret mechanical cues, including cell-cell and cell-substrate interactions, in the microenvironment to collectively regulate various physiological functions. Understanding the influences of these mechanical factors on cell behavior is critical for fundamental cell biology and for the development of novel strategies in regenerative medicine. Here, we demonstrate plasma lithography patterning on elastomeric substrates for elucidating the influences of mechanical cues on neuronal differentiation and neuritogenesis. The neuroblastoma cells form neuronal spheres on plasma-treated regions, which geometrically confine the cells over two weeks. The elastic modulus of the elastomer is controlled simultaneously by the crosslinker concentration. The cell-substrate mechanical interactions are also investigated by controlling the size of neuronal spheres with different cell seeding densities. These physical cues are shown to modulate with the formation of focal adhesions, neurite outgrowth, and the morphology of neuroblastoma. By systematic adjustment of these cues, along with computational biomechanical analysis, we demonstrate the interrelated mechanoregulatory effects of substrate elasticity and cell size. Taken together, our results reveal that the neuronal differentiation and neuritogenesis of neuroblastoma cells are collectively regulated via the cell-substrate mechanical interactions.
doi:10.1038/srep06965
PMCID: PMC4223667  PMID: 25376886
5.  Detection of mRNA in living cells by double-stranded locked nucleic acid probes 
The Analyst  2013;138(17):4777-4785.
Double-stranded probes are homogeneous biosensors for rapid detection of specific nucleotide sequences. These double-stranded probes have been applied in various molecular sensing applications, such as real-time polymerase chain reaction and detection of bacterial 16S rRNA. In this study, we present the design and optimization of double-stranded probes for single-cell gene expression analysis in living cells. With alternating DNA/LNA monomers for optimizing the stability and specificity, we show that the probe is stable in living cells for over 72 hours post-transfection and is capable of detecting changes in gene expression induced by external stimuli. The probes can be delivered to a large number of cells simultaneously by cationic liposomal transfection or to individual cells selectively by photothermal delivery. We also demonstrate that the probe quantifies intracellular mRNA in living cells through the use of an equilibrium analysis. With its effectiveness and performance, the double-stranded probe represents a broadly applicable approach for large-scale single-cell gene expression analysis toward numerous biomedical applications, such as systems biology, cancer, and drug screening.
doi:10.1039/c3an00722g
PMCID: PMC3736730  PMID: 23772441
6.  Molecular Detection of Bacterial Pathogens Using Microparticle Enhanced Double-Stranded DNA Probes 
Analytical chemistry  2011;83(16):6349-6354.
Rapid, specific, and sensitive detection of bacterial pathogens is essential toward clinical management of infectious diseases. Traditional approaches for pathogen detection, however, often require time-intensive bacterial culture and amplification procedures. Herein, a microparticle enhanced double-stranded DNA probe is demonstrated for rapid species-specific detection of bacterial 16S rRNA. In this molecular assay, the binding of the target sequence to the fluorophore conjugated probe thermodynamically displaces the quencher probe and allows the fluorophore to fluoresce. By incorporation of streptavidin-coated microparticles to localize the biotinylated probes, the sensitivity of the assay can be improved by 3 orders of magnitude. The limit of detection of the assay is as few as eight bacteria without target amplification and is highly specific against other common pathogens. Its applicability toward clinical diagnostics is demonstrated by directly identifying bacterial pathogens in urine samples from patients with urinary tract infections.
doi:10.1021/ac2012575
PMCID: PMC4104485  PMID: 21718053
7.  Advances and challenges in biosensor-based diagnosis of infectious diseases 
Rapid diagnosis of infectious diseases and timely initiation of appropriate treatment are critical determinants that promote optimal clinical outcomes and general public health. Conventional in vitro diagnostics for infectious diseases are time-consuming and require centralized laboratories, experienced personnel and bulky equipment. Recent advances in biosensor technologies have potential to deliver point-of-care diagnostics that match or surpass conventional standards in regards to time, accuracy and cost. Broadly classified as either label-free or labeled, modern biosensors exploit micro- and nanofabrication technologies and diverse sensing strategies including optical, electrical and mechanical transducers. Despite clinical need, translation of biosensors from research laboratories to clinical applications has remained limited to a few notable examples, such as the glucose sensor. Challenges to be overcome include sample preparation, matrix effects and system integration. We review the advances of biosensors for infectious disease diagnostics and discuss the critical challenges that need to be overcome in order to implement integrated diagnostic biosensors in real world settings.
doi:10.1586/14737159.2014.888313
PMCID: PMC4104499  PMID: 24524681
iosensor; infectious diseases; matrix effects; microfluidics; sample preparation; system integration
8.  Electrokinetic Focusing and Separation of Mammalian Cells in Conductive Biological Fluids 
The Analyst  2012;137(22):5215-5221.
Active manipulation of cells, such as trapping, focusing, and isolation, is essential for various bioanalytical applications. Herein, we report a hybrid electrokinetic technique for manipulating mammalian cells in physiological fluids. This technique applies a combination of negative dielectrophoretic force and hydrodynamic drag force induced by electrohydrodynamics, which is effective in conductive biological fluids. With a three-electrode configuration, the stable equilibrium positions of cells can be adjusted for separation and focusing applications. Cancer cells and white blood cells can be positioned and isolated into specific locations in the microchannel under both static and dynamic flow conditions. To investigate the sensitivity of the hybrid electrokinetic process, AC voltage, frequency, and bias dependences of the cell velocity were studied systematically. The applicability of the hybrid electrokinetic technique for manipulating cells in physiological samples is demonstrated by continuous focusing human breast adenocarcinoma spiked in urine, buffy coats, and processed blood samples with 98% capture efficiency.
doi:10.1039/c2an35707k
PMCID: PMC4086461  PMID: 22937529
9.  AC Electrokinetics Facilitated Biosensor Cassette for Rapid Pathogen Identification 
The Analyst  2013;138(13):3660-3666.
To develop a portable point-of-care system based on biosensors for common infectious diseases such as urinary tract infection, the sensing process needs to be implemented within an enclosed fluidic system. On chip sample preparation of clinical samples remains a significant obstacle to achieve robust sensor performance. Herein AC electrokinetics is applied in an electrochemical biosensor cassette to enhance molecular convection and hybridization efficiency though electrokinetic induced fluid motion and Joule heating induced temperature elevation. Using E. coli as an exemplary pathogen, we determined the optimal electrokinetic parameters for detecting bacterial 16S rRNA in the biosensor cassette based on the current output, signal-to-noise ratio, and limit of detection. In addition, a panel of six probe sets targeting common uropathogenic bacteria was demonstrated. The optimized parameters were also validated using patient-derived clinical urine samples. The effectiveness of electrokinetic for on chip sample preparation will facilitate the implementation of point-of-care diagnosis of urinary tract infection in the future.
doi:10.1039/c3an00259d
PMCID: PMC3709570  PMID: 23626988
10.  Hybrid electrokinetic manipulation in high-conductivity media† 
Lab on a chip  2011;11(10):1770-1775.
This study reports a hybrid electrokinetic technique for label-free manipulation of pathogenic bacteria in biological samples toward medical diagnostic applications. While most electrokinetic techniques only function in low-conductivity buffers, hybrid electrokinetics enables effective operation in high-conductivity samples, such as physiological fluids (~1 S m−1). The hybrid electrokinetic technique combines short-range electrophoresis and dielectrophoresis, and long-range AC electrothermal flow to improve its effectiveness. The major technical hurdle of electrode instability for manipulating high conductivity samples is tackled by using a Ti–Au–Ti sandwich electrode and a 3-parallel-electrode configuration is designed for continuous isolation of bacteria. The device operates directly with biological samples including urine and buffy coats. We show that pathogenic bacteria and biowarfare agents can be concentrated for over 3 orders of magnitude using hybrid electrokinetics.
doi:10.1039/c1lc20054b
PMCID: PMC4084846  PMID: 21487576
11.  A Biosensor Platform for Rapid Antimicrobial Susceptibility Testing Directly From Clinical Samples 
The Journal of urology  2010;185(1):148-153.
Purpose
A significant barrier to efficient antibiotic management of infection is that the standard diagnostic methodologies do not provide results at the point of care. The delays between sample collection and bacterial culture and antibiotic susceptibility reporting have led to empirical use of antibiotics, contributing to the emergence of drug resistant pathogens. As a key step toward the development of a point of care device for determining the antibiotic susceptibility of urinary tract pathogens, we report on a biosensor based antimicrobial susceptibility test.
Materials and Methods
For assay development bacteria were cultured with or without antibiotics, and growth was quantitated by determining viable counts and electrochemical biosensor measurement of bacterial 16S rRNA. To determine antibiotic susceptibility directly from patient samples, urine was cultured on antibiotic plates for 2.5 hours and growth was determined by electrochemical measurement of bacterial 16S rRNA. For assay validation 252 urine samples were collected from patients at the Spinal Cord Injury Service at Veterans Affairs Palo Alto Health Care System. The biosensor based antimicrobial susceptibility test was completed for samples containing gram-negative organisms. Pathogen identification and antibiotic susceptibility results were compared between our assay and standard microbiological analysis.
Results
A direct biosensor quantitation of bacterial 16S rRNA can be used to monitor bacterial growth for a biosensor based antimicrobial susceptibility test. Clinical validation of a biosensor based antimicrobial susceptibility test with patient urine samples demonstrated that this test was 94% accurate in 368 pathogen-antibiotic tests compared to standard microbiological analysis.
Conclusions
This biosensor based antimicrobial susceptibility test, in concert with our previously described pathogen identification assay, can provide culture and susceptibility information directly from a urine sample within 3.5 hours.
doi:10.1016/j.juro.2010.09.022
PMCID: PMC4051414  PMID: 21074208
urinary tract infections; biosensing techniques; microbial sensitivity tests; point-of-care systems
12.  Intelligent Systems and Technology for Integrative and Predictive Medicine: An ACP Approach 
One of the principal goals in medicine is to determine and implement the best treatment for patients through fastidious estimation of the effects and benefits of therapeutic procedures. The inherent complexities of physiological and pathological networks that span across orders of magnitude in time and length scales, however, represent fundamental hurdles in determining effective treatments for patients. Here we argue for a new approach, called ACP-based approach that combines artificial (societies), computational (experiments) and parallel (execution)methods in intelligent systems and technology for integrative and predictive medicine, or more general, precision medicine and smart health management. The advent of artificial societies that collect the clinically relevant information in prognostics and therapeutics provides a promising platform for organizing and experimenting complex physiological systems toward integrative medicine. The ability of computational experiments to analyze distinct, interactive systems such as the host mechanisms, pathological pathways, therapeutic strategies as well as other factors using the artificial systems will enable control and management through parallel execution of real and arficial systems concurrently within the integrative medicine context. The development of this framework in integrative medicine fueled by close collaborations between physicians, engineers, and scientists will result in preventive and predictive practices of personal, proactive, and precision nature, including rational combinatorial treatments, adaptive therapeutics, and patient-oriented disease management.
PMCID: PMC4039638  PMID: 24883228
13.  Mechanically Induced Intercellular Calcium Communication in Confined Endothelial Structures 
Biomaterials  2013;34(8):2049-2056.
Calcium signaling in the diverse vascular structures is regulated by a wide range of mechanical and biochemical factors to maintain essential physiological functions of the vasculature. To properly transmit information, the intercellular calcium communication mechanism must be robust against various conditions in the cellular microenvironment. Using plasma lithography geometric confinement, we investigate mechanically induced calcium wave propagation in networks of human umbilical vein endothelial cells organized. Endothelial cell networks with confined architectures were stimulated at the single cell level, including using capacitive force probes. Calcium wave propagation in the network was observed using fluorescence calcium imaging. We show that mechanically induced calcium signaling in the endothelial networks is dynamically regulated against a wide range of probing forces and repeated stimulations. The calcium wave is able to propagate consistently in various dimensions from monolayers to individual cell chains, and in different topologies from linear patterns to cell junctions. Our results reveal that calcium signaling provides a robust mechanism for cell-cell communication in networks of endothelial cells despite the diversity of the microenvironmental inputs and complexity of vascular structures.
doi:10.1016/j.biomaterials.2012.11.060
PMCID: PMC3542404  PMID: 23267827
plasma lithography; micropatterning; calcium; endothelial cell; cell signaling
14.  Calcium Wave Propagation in Networks of Endothelial Cells: Model-based Theoretical and Experimental Study 
PLoS Computational Biology  2012;8(12):e1002847.
In this paper, we present a combined theoretical and experimental study of the propagation of calcium signals in multicellular structures composed of human endothelial cells. We consider multicellular structures composed of a single chain of cells as well as a chain of cells with a side branch, namely a “T” structure. In the experiments, we investigate the result of applying mechano-stimulation to induce signaling in the form of calcium waves along the chain and the effect of single and dual stimulation of the multicellular structure. The experimental results provide evidence of an effect of architecture on the propagation of calcium waves. Simulations based on a model of calcium-induced calcium release and cell-to-cell diffusion through gap junctions shows that the propagation of calcium waves is dependent upon the competition between intracellular calcium regulation and architecture-dependent intercellular diffusion.
Author Summary
Calcium wave signal has been found in a wide variety of cell types. Over the last years, a large number of calcium experiments have shown that calcium signal is not only an intracellular regulator but is also able to be transmitted to surrounding cells as intercellular signal. This paper focuses on the development of an approach with complementary integration of theoretical and experimental methods for studying the multi-level interactions in multicellular architectures and their effect on collective cell dynamic behavior. We describe new types of higher-order (across structure) behaviors arising from lower-order (within cells) phenomena, and make predictions concerning the mechanisms underlying the dynamics of multicellular biological systems. The theoretical approach describes numerically the dynamics of non-linear behavior of calcium-based signaling in model networks of cells. Microengineered, geometrically constrained networks of human umbilical vein endothelial cells (HUVEC) serve as platforms to arbitrate the theoretical predictions in terms of the effect of network topology on the spatiotemporal characteristics of emerging calcium signals.
doi:10.1371/journal.pcbi.1002847
PMCID: PMC3531288  PMID: 23300426
15.  Biosensor diagnosis of urinary tract infections: a path to better treatment? 
Urinary tract infection (UTI) is among the most common bacterial infections and poses a significant healthcare burden. The standard culture-based diagnosis of UTI has a typical delay of two to three days. In the absence of definitive microbiological diagnosis at the point of care, physicians frequently initiate empirical broad-spectrum antibiotic treatment, which has contributed to the emergence of resistant pathogens. Biosensors are emerging as a powerful diagnostic platform for infectious diseases. Similar to how blood glucose sensors revolutionized the management of diabetes and pregnancy tests are now conducted at home, biosensors are poised to significantly improve UTI diagnosis. Biosensors are amenable to integration with microfluidic technology for point-of-care applications. This review focuses on promising biosensor technology for UTI diagnosis, including pathogen identification and antimicrobial susceptibility testing and hurdles in the translation of biosensor technology from bench to bedside.
doi:10.1016/j.tips.2011.03.001
PMCID: PMC3106133  PMID: 21458868
16.  Probing cell migration in confined environments by plasma lithography 
Biomaterials  2010;32(7):1848-1855.
Cellular processes are regulated by various mechanical and physical factors in their local microenvironment such as geometric confinements, cell-substrate interactions, and cell-cell contact. Systematic elucidation of these regulatory mechanisms is crucial for fundamental understanding of cell biology and for rational design of biomedical devices and regenerative medicine. Here, we report a generally applicable plasma lithography technique, which performs selective surface functionalization on large substrate areas, for achieving long-term, stable confinements with length scales from 100 nm to 1 cm toward the investigation of cell-microenvironment interactions. In particular, we applied plasma lithography for cellular confinement of neuroblastomas, myoblasts, endothelial cells, and mammary gland epithelial cells, and examined the motion of mouse embryonic fibroblasts in directionality-confined environments for studying the effect of confinements on migratory behavior. In conjunction with live cell imaging, the distance traveled, velocity, and angular motion of individual cells and collective cell migration behaviors were measured in confined environments with dimensions comparable to a cell. A critical length scale that a cell could conceivably occupy and migrate to was also identified by investigating the behaviors of cells using confined environments with subcellular length scales.
doi:10.1016/j.biomaterials.2010.11.009
PMCID: PMC3023939  PMID: 21134692
17.  Electrothermal Fluid Manipulation of High-Conductivity Samples for Laboratory Automation Applications 
JALA (Charlottesville, Va.)  2010;15(6):426-432.
Electrothermal flow is a promising technique in microfluidic manipulation toward laboratory automation applications, such as clinical diagnostics and high throughput drug screening. Despite the potential of electrothermal flow in biomedical applications, relative little is known about electrothermal manipulation of highly conductive samples, such as physiological fluids and buffer solutions. In this study, the characteristics and challenges of electrothermal manipulation of fluid samples with different conductivities were investigated systematically. Electrothermal flow was shown to create fluid motion for samples with a wide range of conductivity when the driving frequency was above 100 kHz. For samples with low conductivities (below 1 S/m), the characteristics of the electrothermal fluid motions were in quantitative agreement with the theory. For samples with high conductivities (above 1 S/m), the fluid motion appeared to deviate from the model as a result of potential electrochemical reactions and other electrothermal effects. These effects should be taken into consideration for electrothermal manipulation of biological samples with high conductivities. This study will provide insights in designing microfluidic devices for electrokinetic manipulation of biological samples toward laboratory automation applications in the future.
doi:10.1016/j.jala.2010.05.004
PMCID: PMC3003926  PMID: 21180401
18.  Clinical Validation of Integrated Nucleic Acid and Protein Detection on an Electrochemical Biosensor Array for Urinary Tract Infection Diagnosis 
PLoS ONE  2011;6(10):e26846.
Background
Urinary tract infection (UTI) is a common infection that poses a substantial healthcare burden, yet its definitive diagnosis can be challenging. There is a need for a rapid, sensitive and reliable analytical method that could allow early detection of UTI and reduce unnecessary antibiotics. Pathogen identification along with quantitative detection of lactoferrin, a measure of pyuria, may provide useful information towards the overall diagnosis of UTI. Here, we report an integrated biosensor platform capable of simultaneous pathogen identification and detection of urinary biomarker that could aid the effectiveness of the treatment and clinical management.
Methodology/Principal Findings
The integrated pathogen 16S rRNA and host lactoferrin detection using the biosensor array was performed on 113 clinical urine samples collected from patients at risk for complicated UTI. For pathogen detection, the biosensor used sandwich hybridization of capture and detector oligonucleotides to the target analyte, bacterial 16S rRNA. For detection of the protein biomarker, the biosensor used an analogous electrochemical sandwich assay based on capture and detector antibodies. For this assay, a set of oligonucleotide probes optimized for hybridization at 37°C to facilitate integration with the immunoassay was developed. This probe set targeted common uropathogens including E. coli, P. mirabilis, P. aeruginosa and Enterococcus spp. as well as less common uropathogens including Serratia, Providencia, Morganella and Staphylococcus spp. The biosensor assay for pathogen detection had a specificity of 97% and a sensitivity of 89%. A significant correlation was found between LTF concentration measured by the biosensor and WBC and leukocyte esterase (p<0.001 for both).
Conclusion/Significance
We successfully demonstrate simultaneous detection of nucleic acid and host immune marker on a single biosensor array in clinical samples. This platform can be used for multiplexed detection of nucleic acid and protein as the next generation of urinary tract infection diagnostics.
doi:10.1371/journal.pone.0026846
PMCID: PMC3204982  PMID: 22066011
19.  Electrochemical Immunosensor Detection of Urinary Lactoferrin in Clinical Samples for Urinary Tract Infection Diagnosis 
Biosensors & bioelectronics  2010;26(2):649-654.
Urine is the most abundant and easily accessible of all body fluids and provides an ideal route for non-invasive diagnosis of human diseases, particularly of the urinary tract. Electrochemical biosensors are well suited for urinary diagnostics due to their excellent sensitivity, low cost, and ability to detect a wide variety of target molecules including nucleic acids and protein biomarkers. We report the development of an electrochemical immunosensor for direct detection of the urinary tract infection (UTI) biomarker lactoferrin from infected clinical samples. An electrochemical biosensor array with alkanethiolate self-assembled monolayer (SAM) was used. Electrochemical impedance spectroscopy was used to characterize the mixed SAM, consisted of 11-mercaptoundecanoic acid and 6-mercapto-1-hexanol. A sandwich amperometric immunoassay was developed for detection of lactoferrin from urine, with a detection limit of 145 pg/ml. We validated lactoferrin as a biomarker of pyuria (presence of white blood cells in urine), an important hallmark of UTI, in 111 patient-derived urine samples. Finally, we demonstrated multiplex detection of urinary pathogens and lactoferrin through simultaneous detection of bacterial nucleic acid (16S rRNA) and host immune-response protein (lactoferrin) on a single sensor array. Our results represent first integrated sensor platform capable of quantitative pathogen identification and measurement of host immune response, potentially providing clinical diagnosis that is not only more expeditious but more informative than the current standard.
doi:10.1016/j.bios.2010.07.002
PMCID: PMC2946447  PMID: 20667707
Electrochemical biosensor; Amperometry; Urinary diagnostics; Urinary tract infections; Biomarkers
20.  Correction: Statistical Metamodeling for Revealing Synergistic Antimicrobial Interactions 
PLoS ONE  2011;6(7):10.1371/annotation/d598d976-2604-429b-a76f-14aeca628a8e.
doi:10.1371/annotation/d598d976-2604-429b-a76f-14aeca628a8e
PMCID: PMC3128627
21.  System Integration - A Major Step toward Lab on a Chip 
Microfluidics holds great promise to revolutionize various areas of biological engineering, such as single cell analysis, environmental monitoring, regenerative medicine, and point-of-care diagnostics. Despite the fact that intensive efforts have been devoted into the field in the past decades, microfluidics has not yet been adopted widely. It is increasingly realized that an effective system integration strategy that is low cost and broadly applicable to various biological engineering situations is required to fully realize the potential of microfluidics. In this article, we review several promising system integration approaches for microfluidics and discuss their advantages, limitations, and applications. Future advancements of these microfluidic strategies will lead toward translational lab-on-a-chip systems for a wide spectrum of biological engineering applications.
doi:10.1186/1754-1611-5-6
PMCID: PMC3117764  PMID: 21612614
22.  Computational simulation of a magnetic microactuator for tissue engineering applications 
Biomedical microdevices  2009;11(6):1259-1267.
The next generation of tissue engineered constructs (TECs) requires the incorporation of a controllable and optimized microstructure if they are to chemically, mechanically, and biologically mimic tissue function. In order to obtain TECs with optimized microstructures, a combination of spatiotemporally regulated mechanical and biochemical stimuli is necessary during the formation of the construct. While numerous efforts have been made to create functional tissue constructs, there are few techniques available to stimulate TECs in a localized manner. We herein describe the design of a microdevice which can stimulate TECs in a localized, inhomogeneous, and predefined anisotropic fashion using ferromagnetically doped polydimethylsiloxane microflaps (MFs). Specifically, a sequential magneto-structural finite element model of the proposed microdevice is constructed and utilized to understand how changes in magnetic and geometrical properties of the device affect MF deflection. Our study indicates that a relatively small density of ferromagnetic material is required to result in adequate force and MF defection (175 μm ~7% TEC strain). We also demonstrate that MF to magnet distance is more important than inherent MF magnetic permeability in determining resulting MF deflection. An experimental validation test setup was used to validate the computational solutions. The comparison shows reasonable agreement indicating a 5.9% difference between experimentally measured and computationally predicted MF displacement. Correspondingly, an apparatus with two MFs and two magnets has been made and is currently undergoing construct testing. The current study presents the design of a novel magnetic microactuator for tissue engineering applications. The computational results reported here will form the foundation in the design and optimization of a functional microdevice with multiple MFs and magnets capable of stimulating TECs in nonhomogenous and preferred directions with relevant spatial resolution.
doi:10.1007/s10544-009-9345-1
PMCID: PMC3075946  PMID: 19685189
Tissue engineering; Biomechanics; Magnetic; Microactuator; Finite element
23.  Rapid Antimicrobial Susceptibility Testing Using High Surface-to-Volume Ratio Microchannels 
Analytical chemistry  2010;82(3):1012.
This study reports the use of microfluidics, which intrinsically has a large surface-to-volume ratio, toward rapid antimicrobial susceptibility testing at the point of care. By observing the growth of uropathogenic E. coli in gas permeable polymeric microchannels with different dimensions, we demonstrate that the large surface-to-volume ratio of microfluidic systems facilitates rapid growth of bacteria. For microchannels with 250 micrometer or less in depth, the effective oxygenation can sustain the growth of E. coli to over 109 cfu/ml without external agitation or oxygenation, which eliminates the requirement of bulky instrumentation and facilitates rapid bacterial growth for antimicrobial susceptibility testing at the point of care. The applicability of microfluidic rapid antimicrobial susceptibility testing is demonstrated in culture media and in urine with clinical bacterial isolates that have different antimicrobial resistance profiles. The antimicrobial resistance pattern can be determined as rapidly as 2 hours compared to days in standard clinical procedures facilitating diagnostics at the point of care.
doi:10.1021/ac9022764
PMCID: PMC2821038  PMID: 20055494
24.  A Microfluidic Cartridge System for Multiplexed Clinical Analysis 
JALA (Charlottesville, Va.)  2009;14(6):407-412.
Cartridge-based microfluidics is a promising technology for clinical diagnostics. By miniaturizing the fluid-handling processes required for genomic and proteomic analyses, reagent and specimen volume is minimized along with the size of the system. We demonstrate an automated microfluidic system capable of performing six multiplexed genomic and proteomic analyses simultaneously, by means of an integrated electrochemical sensor and embedded controls.
doi:10.1016/j.jala.2009.05.002
PMCID: PMC2808045  PMID: 20161584
microfluidics; electrochemical sensor; multiplexed assay; quantitative; molecular analysis; point of care; clinical diagnostics
25.  Statistical Metamodeling for Revealing Synergistic Antimicrobial Interactions 
PLoS ONE  2010;5(11):e15472.
Many bacterial pathogens are becoming drug resistant faster than we can develop new antimicrobials. To address this threat in public health, a metamodel antimicrobial cocktail optimization (MACO) scheme is demonstrated for rapid screening of potent antibiotic cocktails using uropathogenic clinical isolates as model systems. With the MACO scheme, only 18 parallel trials were required to determine a potent antimicrobial cocktail out of hundreds of possible combinations. In particular, trimethoprim and gentamicin were identified to work synergistically for inhibiting the bacterial growth. Sensitivity analysis indicated gentamicin functions as a synergist for trimethoprim, and reduces its minimum inhibitory concentration for 40-fold. Validation study also confirmed that the trimethoprim-gentamicin synergistic cocktail effectively inhibited the growths of multiple strains of uropathogenic clinical isolates. With its effectiveness and simplicity, the MACO scheme possesses the potential to serve as a generic platform for identifying synergistic antimicrobial cocktails toward management of bacterial infection in the future.
doi:10.1371/journal.pone.0015472
PMCID: PMC2988685  PMID: 21124958

Results 1-25 (29)